title: Opening windows into the cell: focused-ion-beam milling for cryo-electron tomography.
abstract: Cryo-electron tomography (CET) is ideally suited for bridging the resolution gap between molecular and cellular structural studies. However, CET is limited to a sample thickness under 500nm, which is thinner than most cells. Here, we review a method for preparing cells for CET using focused-ion-beam milling, a technique commonly used in materials science. Adapted to cryogenic conditions, FIB milling can be applied to various cell types to produce samples thin enough for CET that do not present the artefacts typical to other preparation techniques, for example, cryo-ultramicrotomy, effectively opening windows into intact cells. Samples can be produced routinely and reproducibly. The data obtained from CET can be used for structural studies in situ, or to do quantitative cell biology studies, in which cells can be observed at the molecular level under different physiological conditions.
authors.affiliation: Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried 82152, Germany.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Villa
authors.forename: Miroslava
authors.initials: M
authors.lastname: Schaffer
authors.forename: Jürgen M
authors.initials: JM
authors.lastname: Plitzko
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Baumeister
mesh_terms: D002477:Cells / Q000648:ultrastructure*; D020285:Cryoelectron Microscopy / Q000379:methods*; D055032:Electron Microscope Tomography / Q000379:methods*